Mass spectrometry has become the method of choice for fast and efficient identification of proteins in biological samples. Tandem mass spectrometry of peptides in a complex protein mixture can be used to identify and quantify the proteins present in the original mixture. Tandem mass spectrometers achieve this by selecting single m/z values and subjecting the precursor ions to fragmentation, providing product ions that can be used to sequence and identify peptides. The information created by the product ions of a peptide can be used to search peptide and nucleotide sequence databases to identify the amino acid sequence represented by the spectrum and thus identify the protein from which the peptide was derived. To identify peptides, database searching programs typically compare each MS/MS spectrum against amino acid sequences in the database, and a probability score is assigned to rank the most likely peptide match. The algorithms typically utilize mass-to-charge ratio (m/z) information for identification purposes of the various product ions.
Fragmentation can be provided by various methodologies and mechanisms. Ion activation techniques that involve excitation of protonated or multiply protonated peptides, include collision-induced dissociation (CID), and infrared multiphoton dissociation (IRMPD) for example, and have been used to identify sequences. In these dissociation methods translational energy is imparted to the peptide and is converted into vibrational energy that is then distributed throughout the bonds of the peptide. When the energy imparted to a particular bond exceeds that required to break the bond, fragmentation occurs and product ions are formed. The cleavage may not always however, occur along the backbone of the peptide if, for example, the side-chain of the peptide has elements that inhibit cleavage along the backbone, by providing a lower energy pathway and cleavage site on a side-chain. This preferential cleavage of the side-chain bonds rather than the polypeptide bonds often results in the provision of information primarily about the side-chain sequences and not the peptide sequence.
Other mechanisms of fragmentation include for example, those in which the capture of a thermal electron is exothermic and causes the peptide backbone to fragment by a non-ergodic process, those that do not involve intramolecular vibrational energy redistribution. Such methodologies include Electron Capture Dissociation (ECD) and Electron Transfer Dissociation (ETD). ECD and ETD occur on a time scale that is short compared with the internal energy distribution that occurs in the CID process, and consequently, most sequence specific fragment forming bond dissociations are typically randomly along the peptide backbone, and not of the side-chains.
Though non-ergodic reactions such as ETD or ECD fragmentation appear to offer the best solutions for peptide determination, these techniques create their own problems. ECD can not be performed with trap-type mass analyzers since the electrons created by the reaction do not typically retain their thermal energy long enough to be trapped, thus ECD is typically performed on a FT-ICT mass spectrometer. These instruments are expensive. ETD fragmentation particularly of large peptides and proteins, which can be performed by an ion trap, often leads to spectra too complicated for direct interpretation. Typically, these larger peptides are highly charged, and their fragment ions are similarly multiply charged, with charge states of +2, +3, +4, +5, +6 and even +7 observed. The limited m/z resolution of currently available mass analyzers makes interpretation of these highly charged product m/z spectral data difficult. In addition, the charge state determination is more complicated and important than for CID where normally charge states up to only +4 are observed.
A precursor subjected to the ETD fragmentation process fragments mainly along its backbone, generating predominantly fragments of the precursor ion. However, in addition to the fragment ions, peaks are generally seen for ions which have been subjected to neutral loss, such as water (−18 Da) for example. Ions from side chain cleavage are generally not observed. Despite the absence of side chain cleavage, the spectral data obtained via the ETD process is typically possesses spectral information that may contain little or no “useful” information in terms of peptide sequencing or identification.
For large peptides and proteins, and the large number of possible charge states, the number of possible matches in a database is also larger. For example, if the precursor ion has a charge state of +3, each fragment of the precursor found in the MS/MS or MSN spectral data can have a possible charge state of +3, +2 or +1. Since it is not possible to directly determine the charge state of each of the fragments in a MS/MS spectrum (the spectrum only provides mass to charge ratio information), if the precursor ion is not known, several searches must be performed. In this case, separate searches considering possible +3, +2 and +1 precursor ion charge states may need to be performed. This is consuming in terms of time and space, in terms, for example, of computer storage space, the number of searches performed, computer execution time, and the valuable time of the scientist in reviewing the data.